Louisiana State University LSU Digital Commons LSU Master's Theses Graduate School 2012 The effect of frozen storage on the survival of probiotic microorganisms found in traditional and commercial kefir Keely Virginia O'Brien Louisiana State University and Agricultural and Mechanical College, [email protected] Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_theses Part of the Animal Sciences Commons Recommended Citation O'Brien, Keely Virginia, "The effect of frozen storage on the survival of probiotic microorganisms found in traditional and commercial kefir" (2012). LSU Master's Theses. 3319. https://digitalcommons.lsu.edu/gradschool_theses/3319 This Thesis is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Master's Theses by an authorized graduate school editor of LSU Digital Commons. For more information, please contact [email protected]. THE EFFECT OF FROZEN STORAGE ON THE SURVIVAL OF PROBIOTIC MICROORGANISMS FOUND IN TRADITIONAL AND COMMERCIAL KEFIR A Thesis Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Master of Animal, Dairy and Poultry Sciences In The Department of Animal Sciences By Keely Virginia O’Brien B.S., University of Tennessee, Chattanooga 2007 May, 2012 1 DEDICATION This thesis is dedicated to my friends and family (especially the kefir makers, you know who you are….) and to fermentation enthusiasts everywhere! ii ACKNOWLEDGEMENTS I would like to thank my family for their unwavering love and support. Thanks are also owed to Dr. Boeneke, Dr. Aryana and Dr. Prinyawiwatkul, my thesis committee members. I would also like to thank all my professors and friends who made this an exciting and challenging experience. Lastly, I would like to thank all the people who, through a shared love of living, “breathing”, nourishing foods, have influenced me in a way that no traditional classroom ever could; so, to all the backyard farmers, home brewers, chicken huggers and ferment everythingers: thank you, thank you, thank you. You are my inspiration, the spark to my creativity, and, without a doubt, will always be my favorite teachers. iii TABLE OF CONTENTS DEDICATION……………………………………………………….……………………ii ACKNOWLEDGEMNTS………………………………………………………………..iii LIST OF TABLES………………………………………………………………………..v LIST OF FIGURES………………………………………………………………………vi ABSTRACT……………………………………………………………………………..vii CHAPTER 1. INTRODUCTION………………………………………………………...1 1.1 Milk and Fermentation Throughout History…………………………………………..1 1.2 Goat Milk……………………………………………………………………………..2 1.3 Principles of Milk Fermentation……………………………………………………...3 1.4 Kefir Definition and Origins………………………………………………………….4 1.5 Kefir Starter Culture………………………………………………………………….5 1.6 Microflora…………………………………………………………………………….8 1.6.1 Lactobacilli………………………………………………………………………….9 1.6.2 Lactococci…………………………………………………………………………...9 1.6.3 Yeasts………………………………………………………………………………10 1.7 Kefiran………………………………………………………………………………11 1.8 Fermentations and Production of Flavor Compounds………………………………13 1.9 Health Benefits………………………………………………………………………15 1.10 Commercial Manufacture………………………………………………………….17 1.11 Frozen Dairy Products……………………………………………………………..19 CHAPTER 2. MATERIALS AND METHODS………………………………………..22 2.1 Experimental Design………………………………………………………………...22 2.2 Sample Preparation………………………………………………………………….22 2.3 Microbial Enumeration………………………………………………………………24 2.4 Statistical Analysis…………………………………………………………………..25 CHAPTER 3. RESULTS………………………………………………………………..26 3.1 Lactobacilli………………………………………………………………………….28 3.2 Lactococci…………………………………………………………………………...30 3.3 Yeasts………………………………………………………………………………..33 CHAPTER 4. DISCUSSION…………………………………………………………….36 CHAPTER 5. CONCLUSIONS AND FUTURE WORK………………………………41 REFERENCES…………………………………………………………………………..44 VITA……………………………………………………………………………………..52 iv LIST OF TABLES Table 2.1 The effects of type and storage treatments on the reduction rate of microorganisms…………………………………………………………………..………27 Table 2.2 Mean counts (log cfu/ml) of viable lactobacilli following storage periods.....28 Table 2.3 Mean counts (log cfu/ml) of viable lactococci following storage periods..…30 Table 2.4 Mean counts (log cfu/ml) of viable yeasts following storage periods……...33 v LIST OF FIGURES Figure 2.1 Reduction rates of lactobacilli, lactococci and yeast populations in traditional and commercial kefir during 30 days of frozen storage…………………………………27 Figure 2.2 Reduction of lactobacilli in traditional and commercial kefir during 30 days of frozen storage……………...………………………………………………………….29 Figure 2.3 Reduction of lactococci in traditional and commercial kefir during 30 days of frozen storage…………………………………………………………………………….32 Figure 2.4 Reduction of yeasts in traditional and commercial kefir during 30 days of frozen storage…………………………………………………………………………… 34 vi ABSTRACT Kefir is a fermented milk traditionally made from a unique starter culture, which consists of numerous bacteria and yeast species bound together in an exopolysaccharide matrix produced by certain lactic acid bacteria. Many health benefits are associated with traditionally produced kefir; however, bulging and leaking packaging, caused by secondary yeast fermentation during storage, has limited large scale manufacture traditionally produced kefir. Commercial kefir products have been designed to reduce these effects by using a pure starter culture consisting of a mixture of bacteria and yeast species that give a flavor similar to traditional kefir, but some health benefits may be lost in commercial production due to reduced microbial diversity and lack of beneficial exopolysaccharides. In this study, traditional and commercial kefir was frozen to study the effects of frozen storage on the viability of probiotic bacteria over time. The traditional kefir was prepared by inoculating 1 L of pasteurized whole goats milk with approximately 30 g of kefir grains. Commercial kefir was prepared by inoculating 1 L of full fat, pasteurized goat milk with a commercial kefir starter. The milk was allowed to ferment at room temperature (24-28°C) until pH 4.6 was reached. Samples were frozen (- 8 to -14°C.) immediately following the completion of fermentation and were thawed and plated for lactobacilli, lactococci and yeasts on day 0, day 7, day 14 and day 30 of frozen storage. Statistical analysis was preformed by statistical analysis software (SAS®) using the variance analysis (ANOVA) f-test, with a confidence interval of 95% (P<0.05). Means were compared by the least significant difference (LSD) test. Lactobacilli, lactococci and yeasts were significantly (P<0.05) reduced in number during frozen storage; however, the traditionally produced kefir was shown to have significantly vii (P<0.05) higher counts of bacteria and yeast at each sampling. It was concluded that frozen storage and the development of frozen kefir products could eliminate most packaging concerns associated with the large scale manufacture of traditionally produced kefir, resulting in increased production and marketability of this healthful product. viii CHAPTER 1. INTRODUCTION 1.1 Milk and Fermentation Throughout History Fermented milks have been a staple food, or present in some amounts, in the diets of many diverse and geographically widespread cultures throughout history. Peoples who were traditionally associated with herding or keeping livestock, be it cattle, sheep, goats, mares or water buffalo, discovered and subsequently refined the process of fermentation as a method of milk preservation; and the types of fermented milks are as varied as the cultures that produce them, ranging from the traditional sour milks of Eastern Europe to the hard salty cheeses developed throughout the Mediterranean region. Production of the first fermented milks dates back to 7000 BC with origins in the middle and far-east of Asia, making it one of the oldest methods of long term food preservation. A further spreading east of these traditions, by way of Russia and Eastern Europe, by the Tartars, Mongols and Huns occurred during their conquests (Vasiljevic, et. al., 2008). The expansion of areas that maintained livestock as a source of meat and dairy food by the introduction of herds and traditional production methods, and subsequent industrialization of dairy food production, has led to a total worldwide domestic ruminant population of nearly three billion at the beginning of the twenty-first century (Weimer, 2001). Although the original fermenters did not take into account the microbiological processes involved, traditions were established that ensured the methodologies and knowledge required to produce the flavors and textures associated with these products were kept 1 intact. Often, these practices were passed down from generation to generation within local communities, feudal states and monasteries (Caplice, et. al., 1999). Over time, the tastes associated with fermented milk, such as the characteristic acidic flavor, may have also become associated with increased health and longevity, thus furthering its spread and increasing manufacture throughout and within ancient populations. 1.2 Goat Milk The type of milk originally used in the production of fermented foods was determined by the type of milk producing mammal that was nearest to a group of people or the indigenous species that was domesticated in the region. Goat milk contains approximately 4.5% lipids, with the highest amount being medium chain-length